The specific ion transport systems localized in the brain capillary endothelium have been implicated in the regulation of water-electrolyte homeostasis in the brain. This report that hypoxia alters K+ transport systems in cultured rat brain capillary endothelial cells (RBEC). Uptake of 86Rb+ ( a tracer for K+, 2.5 microCi/ml) into RBEC was measured in HEPES-buffered Medium 199 at room temperature for 10 min. Ouabain- sensitive and bumetanide-sensitive K+ uptake was defined as Na+K+-ATPase and Na+K+ Cl- cotransport activity, respectively. Hypoxia (95% N2/5% CO2, 24 hr) reduced (61% of control) the Na+K+-ATPase activity, whereas it increased (149% of control) the Na+K+-cotransport activity (respective control values = 4.6 and 5.5 nmol/mg protein/min, n=8). Oligomycin, a metabolic inhibitor (1 microg/ml), similarly affected both ion transport systems in a time-dependent manner, which caused an increased (133% of control) total K+ uptake. Oligomycin also increased the rate of K+ efflux up to 129% of the control without altering the total intracellular K+ content (values in mu mol/mg protein; control = 96, oligomycin = 0.93). The oligomycin augmented Na+K+Cl- cotransport activity was reduced by the protein tyrosine kinase inhibitors (genistin, 50 microM; herbimycin A, 10 microM) without being affected by an inhibitor of protein kinase C (bisindolylmateimide, 500 nM) or protein kinase A(H8, 20 microM), indicating the involvement of protein-tyrosine phosphorylation. The data indicate that the up-regulated K+ uptake during hypoxia is due to an increased Na+K+Cl- cotransport activity. It is suggested that a similar mechanism may play a role in the disturbance of water-electrolyte homeostasis described in ischemic brain.